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In Vitro Cultivation of Anisakis Simplex: Pepsin Increases Survival and Moulting from Fourth Larval to Adult Stage

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In Vitro Cultivation of Anisakis Simplex: Pepsin Increases Survival and Moulting from Fourth Larval to Adult Stage 285 In vitro cultivation of Anisakis simplex: pepsin increases survival and moulting from fourth larval to adult stage L.IGLESIAS, A.VALERO, R.BENI; TEZ and F.J.ADROHER* Department of Parasitology, Faculty of Pharmacy, University of Granada, 18071-Granada, Spain (Received 28 October 2000; revised 23 March 2001; accepted 23 March 2001) This paper describes the in vitro cultivation of the 3rd-larval stage (L3) of Anisakis simplex to adulthood in a much simpler and easier to prepare medium than those described to date. The adult males obtained are between 3n8 and 6n5 cm long and the females between 4n5 and 8n0 cm. Some individually cultivated females laid eggs which had an average size of 44n4i50n5 µm. The culture conditions were as follows: medium RPMI-1640 supplemented with 20% heat-inactivated fetal bovine serum and 1% commercial pepsin, at pH 4n0 and a temperature of 37 mC, and in air atmosphere with 5% CO#. The pepsin was found to be the key to the success of the culture. The average survival of the worms in the culture increased from 50 to 88 days, due to the fact that the survival of the adults practically doubled (increasing by 1n9 times). Furthermore, the number of worms that completed the 4th moulting (M4) increased by 4n2 times, from 22n9to95n6%. This culture medium may facilitate, due to its simplicity, the study of anisakids, or at least of A. simplex, constituting another step towards achieving a complete in vitro life-cycle for these parasites. Key words: Anisakiosis, Anisakis simplex, in vitro cultivation, pepsin, moulting, survival. (blue whiting), family Gadidae, purchased from the fish market of Granada (Southern Spain). The blue Anisakis simplex is a parasitic nematode of marine whiting, on the Atlantic and Mediterranean Spanish mammals, which infects a large variety of fish, coasts, is frequently parasitized by A. simplex s.l. among other hosts, during its larval stage. These (Ruiz-Valero et al. 1992; Valero et al. 2000). The parasitized fish may be ingested by humans, thus worms, found free in the host body cavity, were releasing the parasite larvae into the stomach, 20 mm or more in length, and were collected with possibly resulting in anisakiosis if the fish were not the help of a needle with a blunt tip, placed on a Petri adequately cooked. dish with 0n9% NaCl solution and washed in it In vitro cultivation of anisakids has had some several times. The worms were observed individually success, but is difficult to carry out due to the fact under an inverted microscope and those which that the culture media used to date are complex as showed any kind of internal or external damage were regards both their composition and their preparation discarded. They were then identified according to (Townsley et al. 1963; Van Banning, 1971; Grabda, morphological features (Hartwich, 1974; Petter & 1976; Carvajal et al. 1981; Likely & Burt, 1989, Maillard, 1988). Over 800 L3 were used in this 1992), study. Forty L3 (ca. 0n5% of total) of A. simplex s.l. Our aim was to develop a culture medium that was from the same hosts wee preserved at k70 mC for more defined in its composition and simpler to electrophoretic identification purposes. These larvae prepare, to make it easier to obtain adult parasites. In were analysed individually by isoenzyme electro- this paper, the defined medium used, supplemented phoresis using thick starch gel in a continuous buffer with fetal bovine serum and pepsin, allowed in vitro system (Nascetti et al. 1986; Martı!n-Sa! nchez, development of the 3rd larval stage (L3) of Anisakis Paniagua & Valero, 1998; Iglesias, Martı!n-Sa! nchez, simplex s.l. Rudolphi, 1809 to mature adults. Adroher & Valero, unpublished observations). Prior to cultivation, each larva was individually placed in an antibiotic-antifungal solution and axenized as described elsewhere (Iglesias, Valero & Adroher, 1997). Worms were cultured on a sterile The worms selected for our study were 3rd-stage polystyrene 24-well tissue-culture plate. The culture larvae of Anisakis simplex s.l. Rudolphi, 1809 isolated medium (1 ml) was placed into each well with 1 from the host Micromesistius poutassou Risso, 1826 parasite. The culture plates were then placed in an incubator at 37 mC and 5% CO# in humid air (except * Corresponding author: Departamento de Parasitologı!a, when indicated), and the culture medium was Facultad de Farmacia, Universidad de Granada, E-18071 Granada, Spain. Fax: j34 958 243862. E-mail: renewed twice a week. The worms were observed fadroher!ugr.es daily for mobility, moulting and survival. Parasitology (2001), 123, 285–291. " 2001 Cambridge University Press DOI: 10.1017\S0031182001008423 Printed in the United Kingdom L. Iglesias and others 286 The culture medium (RPMI) was RPMI-1640 experiment in which the larvae were subjected to a plus 20% (v\v) heat-inactivated fetal bovine serum change in pH on the 5th day (from pH 7n2topH2n0, (IFBS). To the medium, where indicated, IFBS 3n0 and 4n0), when all the parasites had moulted to (40% v\v), horse blood (1% v\v), pepsin (1% p\v), L4. Moulting to L5 only occurred between days 43 -cysteine (4 m) or glutathione (4 m) were added and 71 in the medium in which the pH was changed and the worms cultivated at several pHs. to 4n0, 5 L5 were produced (20% of the initial larvae; The terms ‘maximum survival’ (Smax), ‘survival 3K,2L) with a SL& of 39n2 days. No statistically 50’ (S&!), and ‘average survival’ (Sav) in culture have significant differences were observed with the control previously been defined by us (Iglesias et al. 1997) as at pH 4n0 from the beginning of the culture (4 L5, follows: Smax is the day the last living nematode in 19% of the initial larvae; 2K and 2L; M4 between the experiment dies. S&! is the day on which 50% of days 54 and 64 of the culture; SL& 31n0 days). Other nematodes in the experiment are dead and Sav is the manipulations of pH produced suboptimal devel- arithmetic mean day of death of each nematode in opment. The high survival rates in these media at the experiment. The data are expressed as the pH 4n0 should be noted. meanpstandard error (..). Pepsin is a component of the gastric juice in the Similarly, other terms are defined as follows: ‘L5- stomachs of mammals. For this reason, we added survival’ (SL&) is the arithmetic mean of the days that pepsin to the culture media at various times during worms survive after the 4th to 5th stage moult (M4). cultivation. In the first assay, at pH 7n2, moulting to ‘L4-survival’ (SL%) is the arithmetic mean of the L5 did not occur with or without pepsin. However, days from M3 to M4. M4av is the arithmetic mean of when the assay was carried out at pH 4n0, there was the culture day on which the M4 of each nematode in a highly significant difference between the medium the experiment is completed. The data are expressed with or without pepsin added from the beginning −' as the meanp.. L4 indicates the larvae that (Sav, P ! 1i10 ; SL&, P ! 0n003). Some moulting completed M3 (i.e. the 3rd to 4th stage moult), and to L5 occurred in all media at pH 4n0 and reached L5 the larvae that completed M4 to adulthood. A 100% in the medium where pepsin was added at the statistical comparison of the culture data was made beginning. This M4 occurred between 20 and 60 using the Student’s t distribution. days of cultivation. The later the pepsin was added The media, sera and reagents were purchased to the culture, the lower the Sav and percentage from Sigma Chemical Co. (St Louis, MO, USA) and moulting to L5 (Table 2). Opposite results occurred Bio-Whittaker (Walkersville, MD, USA). Pepsin when the pepsin was eliminated from (i.e. not added was obtained from Probus (Barcelona, Spain). The to) the culture medium at different culture times commercial pepsin used in our experiments was (Fig. 2). On the other hand, 1 female, grown in the analysed by SDS–PAGE (Laemmli, 1970). Protein medium with pepsin added on the 14th day, began concentration was determined according to the ovoposition on the 97th day of cultivation, 51 days method of Lowry et al. (1951). after moulting to L5. This worm laid 15442 eggs in 16 days (with a maximum of 5672 eggs laid on 1 day). Other changes in the culture medium resulted in Forty L3 of A. simplex s.l. collected from M. reduced moulting and survival times compared with poutassou were electrophoretically identified as A. the pepsin-added media. The addition of either simplex s.s. (39) or A. pegreffii (1) (Nascetti et al. glutathione or -cysteine alone, to the culture 1986). medium inhibited the moult to L5. Even with The greatest moulting percentages to L4 were pepsin, -cysteine prevented the moult to L5 and obtained in the presence of 5% CO#, no matter glutathione significantly reduced the numbers which medium was used. The best parameters both moulting. Similarly, increasing the percentage of for survival and moulting occurred in the nutritive IFBS or adding horse blood alone, inhibited the medium RPMI (Table 1). Consequently, the fol- moult to L5. With pepsin and increased IFBS, the lowing experiments were carried out in the RPMI numbers moulting were reduced. In this experiment, medium in air atmosphere j5% CO#. in the control medium RPMI with pepsin, at pH 4n0, When the culture was carried out at different pHs, 22 worms moulted to L5 (91n6%) between days 22 it was observed that at any pH used, all or almost all and 46, of which 10 were male and 12 female, with a the L3 reach L4, but L5 were only produced at pH SL& of 56n1 days.
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